LOCUS       sequence-450666-        8174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector to express 1-547 aa of Regnase-3 tagged 
            with FLAG at N-term.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8174)
  AUTHORS   Bataclan M, Leoni C, Moro SG, Pecoraro M, Wong EH, Heissmeyer V, 
            Monticelli S
  TITLE     Crosstalk between Regnase-1 and -3 shapes mast cell survival and 
            cytokine expression.
  JOURNAL   Life Sci Alliance. 2024 Jun 3;7(8):e202402784. doi: 
            10.26508/lsa.202402784. Print 2024 Aug.
  PUBMED    38830770
REFERENCE   2  (bases 1 to 8174)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Life Sci 
            Alliance."; date: "2024-06-3"; volume: "7(8):e202402784. doi"; 
            pages: " 10.26508/lsa.202402784. Print 2024 Aug"
COMMENT     Sequence Label: pCDNA3-FLAG-Regnase-3_1-547 aa
FEATURES             Location/Qualifiers
     source          1..8174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     306..325
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    610..731
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(647..666)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     701..720
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(780..796)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(780..796)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(793..815)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    804..820
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(828..834,835..852,853..858))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    873..894
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1011..1028)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1182..1770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1262..1281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1941..2732,2733..2801))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2564..2583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2802..2906)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2981..3000)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3172..3551
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3552..3755
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3706..3726
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3800..3819
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3800..3818
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3881..3890
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3890..3913
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6608..6634
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(6664..6682)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(6665..6682)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(6702..6719)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6708..6932
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6978..7406
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7065..7084)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7275..7296
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7415..7435)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7420..7749
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7600..7735
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7662..7681
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             join(7816..8174,1..436)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7870..7889)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
       61 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
      121 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
      181 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
      241 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
      301 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
      361 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
      421 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagt gacgcccaac
      481 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
      541 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
      601 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
      661 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
      721 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
      781 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
      841 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
      901 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
      961 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     1021 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     1081 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     1141 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1201 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1261 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1321 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1381 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1441 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1501 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     1561 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     1621 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1681 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1741 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1801 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     1861 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     1921 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     1981 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     2041 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     2101 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     2161 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     2221 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     2281 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     2341 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     2401 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     2461 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     2521 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     2581 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     2641 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     2701 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     2761 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     2821 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     2881 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     2941 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     3001 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     3061 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
     3121 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
     3181 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     3241 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     3301 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     3361 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     3421 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     3481 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     3541 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
     3601 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     3661 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     3721 ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg
     3781 cttactggct tatcgaaatt aatacgactc actataggga gacccaagct tggtaccgag
     3841 ctcggatcca ctagtaacgg ccgccagtgt gttcgaattc gccaccatgg attataaaga
     3901 tgatgatgat aaaggctcct ccggcatggc tgcggagaag acattgcagg aatacggggt
     3961 gctttgcatt caagaataca ggaaaagcag caaagtggag tcaagtgcac ggaacagctt
     4021 catgggcctg aaggatcacc tggggcatga tcttggccat ctttatatgg agagcactga
     4081 cccacagatg agtgcagctg tgccttggcc tatggtagag aaaccaacaa tggatacagt
     4141 caattcgggg aaggaaggaa aaggggtgtc agaagagaat gtgagctccg gcgactctga
     4201 gggaagcacg agttctgatc atgagtcaga acaactgagt agcctttcgg tagagccatg
     4261 ctcattaacc aagactcaca gacagctatg tcgctctccc tgcttagagc ctcgcctact
     4321 gaaacacagt gacattttgc aagactttaa accagaagag tcccagactc catctaagga
     4381 agtgaagaaa ccccctgatg tggttcgaga ataccaaaca aaactggagt ttgcacttaa
     4441 gttaggttat tctgaagaac aggtccagct tgtgctaaac aaacttggta ctgatgcttt
     4501 aatcaatgat atattgggag aacttgtcaa acttggaaat aaaagtgaag ccgatcaaac
     4561 agttagtact attaacagcg taatgcgaga aacctcatcc ctggagtctc agaggtctga
     4621 atctccaatg caagaggttg tagtagatga cggtgagaat ctgagaccag tagttattga
     4681 tggcagcaat gtggcaatga gccatggaaa caaagaagta ttctcgtgcc gaggaataaa
     4741 attggcggtg gattggtttt tggaaagagg ccataaagat atcacggtct ttgttcctgc
     4801 ttggagaaaa gaacagtccc gccctgacgc cctcatcaca gatcaggaaa ttttacgtaa
     4861 actagagaag gaaaaaatcc tggtattcac accatcacgg cgtgtccagg gcaggcgagt
     4921 ggtgtgctat gatgacagat tcatcgtgaa gctggctttt gagtccgatg gtataattgt
     4981 gtccaatgat aactacagag atttggcaaa tgagaaacca gaatggaaga agttcataga
     5041 tgagcggtta ttgatgtatt cctttgtcaa tgacaagttc atgcccccag atgaccctct
     5101 tggcagacat ggcccaagtc tggataattt tctgaggaag aaacccattg ttcctgagca
     5161 caaaaagcag ccttgtccct atggaaagaa gtgtacgtat ggacacaagt gcaaatacta
     5221 ccacccagaa aggggcagtc agcctcagcg gtcagtggct gatgaacttc gtgccatgtc
     5281 tagaaacaca gcagccaaaa ctacaaatga agggggactg gtgaaaagca acagtgttcc
     5341 ttgcagcacc aaggcagaca gcacttcgga tgttaaacgc ggtgctccaa agaggcagtc
     5401 agacccaagc ataaggactc acgtctacca agacatagag gagaagcttc ccaccaaaaa
     5461 caagttggaa accaggtctg taccttcctt agttagtatc ccggccactt ctactgcaaa
     5521 accccaaagc actacacctt taagcaatgg ccttccatct ggagtttaag aattcgccac
     5581 catgttccca cctcaggatc aaagaccaca gggacaatac cctccaatga tgatggcaac
     5641 caaaaatcat ggaacgccaa tgccttatga acagtatcca aaatgtgact cccctgttga
     5701 cgttggatat tattccatgt tgaatgcata ttctaatctg agtatctcag gcccccgaag
     5761 tcctgaaagg cgtttctcac tagacactga ttacagagta aactctgtgg cttcagactg
     5821 cagcagtgaa gggagcatga gttgtgggag cagcgactcc tacgtggggt acaatgaccg
     5881 ctcctatgtc agctctcctg acccacagct ggaagagagt ctcaagtgtc agcacatgca
     5941 cccccacagc cgccttaatt cccagccctt cctgcagaat ttccacgatc ccttaaccag
     6001 agtccagagt tacagtcacg aagaaccaaa gttccatccc aagcgtcctc ttccacactt
     6061 ggctatgcat ctgcagcacc ctgccgtggg tgcccgctcc agctgtcctg gcgattaccc
     6121 ctccccacca agttcagcac attcgaaggc accacaccta gggaggtcct tagtggccac
     6181 gagaatagac agcatttctg attcccgact ctatgatagt tctccttcaa gacaaagaaa
     6241 gccttattct cgccaggaag gcctaggaag ctggggtagg ccaagctatg ggctggaagc
     6301 atatggctat cggcagacgt actccttacc cgataattcc acgcccccat gctatgagtc
     6361 gatcaccttc cagagcctgc ctgagcagca ggaacccacc tggagaatcc cctactgtgg
     6421 aatgccacac gatcctccaa ggtaccagga caaccgagag aagattttta tcaatttgtg
     6481 taacatcttc ccccctgacc ttgtgaggct cgtcatgaaa aggaatcctc atatgacgga
     6541 tgcccagcag ctcgccgcag ccatcttagt ggagaaatcg cagctgggtt acggaggctc
     6601 ctccggctat ccgtatgatg tgccggatta tgcgtgatcg agcatgcatc tagagggccc
     6661 tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag
     6721 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     6781 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     6841 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     6901 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     6961 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     7021 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     7081 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     7141 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     7201 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     7261 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     7321 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     7381 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     7441 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     7501 accaggtgtg gaaagtccac aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     7561 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     7621 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     7681 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     7741 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     7801 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     7861 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     7921 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     7981 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     8041 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     8101 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     8161 atggctgatg caat
//
